The Netrin family of proteins were first identified as attractive guidance cues for growing axons. Our recently-published data show they have a corresponding role in vascular guidance. There were three main results. First, Netrin-1, -2 and -4 induce migration, proliferation, and tube formation in multiple endothelial cell lines. Second, knockdown of netrinla during zebrafish embryogenesis inhibits the formation of the para- chordal vessel (PAV), which has recently been shown to be critical for forming the zebrafish lymphatic sys- tem. Third, exogenous Netrins accelerate revascularization and reperfusion of ischemic tissue in two murine disease models. Thus, using zebrafish and mammalian systems, loss-of-function and gain-of-function strat- egies, and in vivo and in vitro assays, we showed that Netrins stimulate angiogenesis. Our more recent preliminary data show that other Netrin-pathway genes (netrin2, dec, uncSb) are also required for zebrafish PAV formation;that UncSb null and endothelial-specific conditional mutant mice have a specific defect in placental angiogenesis;and that Netrins can also influence hematopoietic and mesen- chymal stem cells (HSCs and MSCs) in culture. In this revised proposal we will build on these results. Exploiting the PI and co-Pi's expertise with cell culture, zebrafish, and mouse systems, we will pursue three specific aims in parallel. Each aim addresses a separate role of Netrin function, allowing us to test for both common and divergent mechanisms during differ- ent aspects of angiogenesis. The first aim will define the role of Netrins and Netrin receptors in zebrafish PAV formation. The second aim will define the placental vascular defect in UncSb mice and address the underlying developmental mechanisms. The third aim will define the effects of Netrins on HSCs and MSCs in culture, and further test whether Netrin can direct the homing of these stem cells in vivo, providing a potential mechanism by which exogenous Netrins can promote revascularization in therapeutic models. Relevance to public health. Formation of the vascular system is critical to normal development, and vas- cular remodeling is critical during responses to injury or disease. These studies will provide fundamental in- sight into the role of Netrin signaling in vascular development and endothelial cell biology, and mechanistic insight into its therapeutic potential. The results may have particular relevance to understanding develop- ment of the lymphatic system and the placenta, and to therapies for ischemic injury.